Epithelial chloride channel 3 Flashcards
some papers on discovery of CF gene product
1989
Rommens, Riordan and Kerem
identifying CF gene
CF gene
chromosome 7
250, 000 base pairs
gene product = CFTR
deltaF508 mutation >70%
CFTR has structural homology with what?
ATP binding cassette ABC
superfamily of transport proteins
3 domains of CFTR
2 hydrophobic membrane spanning domains
2 nucleotide binding domains
unique regulatory domain
MSDs of CFTR
each comprised of 6 alpha helices
NBDs od CFTR
bind and hydrolyse ATP
NBD1 - site of deltaF508mutation
Unique R domain of CFTR
phosphorylation site for PKA
Rich et al 1990
CFTR expression correct defective Cl channel regulation in CF airway
1990 - Rich et al explain
transfect CF cells CFTR
normal cAMP activated Cl currents
deltaF508 did not do this
channels induced by CFTR expression similar to SLCCs
Speculation by Rich et al in 1990
CFTR is a SLCC
S9 insect cell line CFTR
no chloride conductance can you know that? mRNA - CFTR to membrane purified CFTR protein mixed with phospholipids to make vesicles CFTR into vesicles plate with 200microns hole drop vesicle into chamber CFTR incorporated into synthetic membrane
1992 Bear
purification and functional reconstitution of CFTR
purified CFTR incorporated into planar lipid bilayers
1992 bear - what was found?
cAMP activated SLCC
what type of channel is CFTR?
low conductance, cAMP activated chloride channel
SLCC
Kartner 1992
deltaF508 CFTR in CF sweat gland
kartner 1992 - what happened to deltaF508 mutated CFTR?
did not localise to apical membrane
CF may be due to absence of Cl channels rather than defective regulation
What did Denning confirm in 1992?
Kartner et al 1992
Denning 1992 - temperature
lowering temperature permits dF508 to localise to apical membrane?
Sheppard 1993
Mutations in CFTR mild-disease form Cl channels with altered pore properties
Sheppard 1993 findings
3 mutations in 1st MSD of CFTR
affects arginine residues
apical cAMP activated SLCC with reduced currents
what did sheppard in 1993 find that might determine Cl conductivity of channel?
arginine residues
Cotten and Welsh 1999
arginine 347 mutations alter pore architecture of CFTR
findings from Cotten and Welsh 1999
Electrostatic interaction between actionic and anionic amino acid residues stabilises channel structure
asp –> arg or vice versa destabilise channel
switch both simultaneously = no problem
Welsh and Smith 1993 - 4 main potential problems of CFTR
protein production
processing - dF508 not get from ER to membrane
regulation - protein in membrane but not activated by cAMP, PKA
conduction - chloride
